Liver specific transcriptional enhancer

ABSTRACT

Recombinant lentiviruses and transfer vectors for transgene delivery as well as methods for gene therapy using such vectors are disclosed. The invention provides a third generation lentiviral packaging system and a set of vectors for producing recombinant lentiviruses, as well as novel tissue specific enhancer and promoter elements useful for optimizing liver specific transgene delivery. The transgene is preferably a blood clotting factor such as human factor IX (hFIX) or human factor VIII (hFVIII) and can be used for treatment of hemophilia.

[0001] This application is a continuation of U.S. application Ser. No.10/145,289, filed May 14, 2002, now U.S. Pat. No. ______, which claimsbenefit of U.S. provisional application Ser. No. 60/291,083, filed May14, 2001, the entire contents of which are incorporated herein byreference.

TECHNICAL FIELD OF THE INVENTION

[0002] The present invention relates to gene therapy. In particular itrelates to vectors for use in the preparation of recombinantlentiviruses and the use of replication-deficient lentiviral vectors todeliver a therapeutic gene to a target tissue of a subject. Suitabletherapeutic genes include genes that encode clotting factors, such asfactor VIII or factor IX, to treat a blood clotting disease such ashemophilia. The invention further relates to enhancers and promotersuseful for tissue-specific gene therapy.

BACKGROUND OF THE INVENTION

[0003] Gene therapy generally relates to the delivery of one or moreheterologous genes to a subject in order to treat a disease. Hemophiliais a genetic disease caused by a deficiency of a blood clotting factor.There are two types of X-linked bleeding disorders, hemophilia A andhemophilia B. In some cases of von Willebrand disease, the most commonbleeding disorder, deficient levels of vWF result in low levels offactor VIII, mimicking hemophilia A. Hemophilia A affects about 17,000people in the US and is caused by a deficiency in factor VIII. Theincidence of hemophilia B is 1 out of 34,500 men, and it is caused by adeficiency in factor IX. Each of these diseases is an excellenttheoretical candidate for gene therapy, as each has a reasonably simplemolecular pathology and should be remediable by the delivery of a singlegene.

[0004] Successful gene therapy for hemophilia requires both tissuespecific expression, to avoid a counterproductive immune response, andsufficiently high levels of expression to generate a therapeuticresponse. Gene therapy directed at quiescent cells of the liver presentsan additional challenge, as Park et al. teach that stable and efficienttransduction of liver cells with a lentiviral vector requires cellproliferation (Park et al., 2000, Nature Genetics 24:49-52). Park et al.further teach that the injection of doses of the lentiviral vectorsufficiently high to reach therapeutic levels of transgene expression inthe liver produces a very high liver toxicity and a high mortality (Parket al., 2000, Blood 96(3):1173-1176).

[0005] There remains a need for successful gene therapy of quiescentcells that results in therapeutically acceptable cell transduction andthat produces a therapeutic amount of protein without toxicity. There isa particular need for a safe and efficient gene therapy for hemophilia.

SUMMARY OF THE INVENTION

[0006] The invention is based on the surprising discovery thatreplication deficient lentiviral vectors can be used to achieve stablegenetic modification of cells in vivo without vector-mediated toxicityand in the absence of target cell proliferation. The invention thusprovides vectors for transgene delivery as well as methods for genetherapy using such vectors. The invention further provides promoters andenhancers useful for optimizing tissue specific transgene delivery.

[0007] The invention provides a lentiviral producer cell comprising afirst nucleotide sequence comprising a gag, a pol, or gag and pol genes;a second nucleotide sequence comprising a heterologous env gene; and athird nucleotide sequence comprising a lentiviral transfer vector thatcomprises a gene that encodes a blood clotting factor operably linked toan expression control sequence; wherein the producer cell lacks afunctional tat gene. In preferred embodiments, the blood clotting factorcomprises human factor IX (hFIX) or human factor VIII (hFVIII). In oneembodiment, the first, second and third nucleotide sequences are stablyintegrated into the genome of the lentiviral producer cell. Preferably,the lentivirus is a human immunodeficiency virus (HIV), such as HIV-1.In a preferred embodiment, the producer cell further comprises a fourthnucleotide sequence that comprises a rev gene, and/or lacks functionalaccessory genes vif, vpr, vpu, vpx and nef, or a combination thereof.

[0008] Typically, the expression control sequence comprises a liverspecific promoter, such as mouse transthyretin (mTTR) promoter, humanalpha-1-antitrypsin promoter (hAAT), human albumin minimal promoter, ahuman factor VIII endogenous promoter, and/or mouse albumin promoter,and/or a liver specific binding site for transcription, such as EBP,DBP, HNF1, HNF3, HNF4, HNF6, or a combination thereof Preferably, theexpression control sequence comprises an Enh1 enhancer (SEQ ID NO: 10)and an mTTR promoter. Also preferred is an expression control sequencecomprising an α-1-microglobulin/bikunin enhancer and a human factor VIIIendogenous promoter (L-F8).

[0009] The invention additionally provides set of lentiviral vectors foruse in a third generation lentiviral packaging system and for productionof lentiviral producer cells and recombination lentiviruses. Theinvention provides a lentiviral transfer vector that comprises anexpression control sequence operably linked to a transgene, wherein theexpression control sequence comprises a novel enhancer element, Enh1(SEQ ID NO: 10), or a novel combination of enhancer and promoterelements, such as L-F8 or Enh1 and mTTR. The transgene is preferably atherapeutic transgene. A preferred transgene is one that encodes a bloodclotting factor, such as human Factor VIII or human Factor IX. Suchtransgenes encoding blood clotting factors can be used in the treatmentof bleeding disorders such as hemophilia. Also provided is a method ofproducing a lentiviral producer cell comprising transforming a cell withthe set of vectors of the invention. The invention further provides amethod of producing a recombinant lentivirus, and a method of deliveringa transgene to a cell.

BRIEF DESCRIPTION OF THE FIGURES

[0010]FIGS. 1A-1H are graphs showing that delivery of LV-hFIX is safeand efficacious. Adult Swiss nude mice received 2×10⁸ TU of LV-HFIX byeither portal vein (closed squares), tail vein (open squares), directintra-splenic (closed triangles) or direct intra-hepatic (opentriangles) administration and serum hFIX levels determined by ELISA overtime (FIG. 1A). Serum levels of sGPT (FIG. 1B), creatinine (FIG. 1C) andalkaline phosphatase (FIG. 1D) were measured in mice from days 1 to 14following either portal vein (closed squares) or tail vein (opensquares) administration of 2×10⁸ TU of LV-HFIX. This analysis wasexpanded to include the measurement of serum toxicological markers inSwiss nude mice following administration of 2×10⁸ TU of LV-HFIX byeither portal vein (closed squares), tail vein (open squares), directintra-splenic (closed triangles) or direct intra-hepatic (opentriangles) routes: sGPT (FIG. 1E), creatinine (FIG. 1F), alkalinephosphatase (FIG. 1G) and albumin (FIG. 1H). In all figures, the valuesfor the PBS are denoted by the open diamonds.

[0011]FIGS. 2A-2C are graphs showing delivery of LV-hFIX inimmunocompetent mice. Adult Swiss nude (FIG. 2A) and C57Bl/6 (FIG. 2B)mice received 2×10⁸ TU of LV-hFIX by either portal vein (closed squares)or tail vein (open squares) administration and serum hFIX levelsdetermined by ELISA over time. FIG. 2C: Levels of anti-hFIX antibodieswere measured in C57Bl/6 following LV-hFIX vector administration.

[0012]FIGS. 3A-3B are bar graphs showing biodistribution andquantification of lentiviral genomes in LV-hFIX mice. Groups of 4 adultSwiss nude mice received 2×10⁸ TU of LV-hFIX by either portal vein(closed bars) or tail vein (open bars) and 48 days after gene transfer,the following tissues were collected: liver, lung, kidney, heart,spleen, brain, pancreas, duodenum, testis and lymph node. DNA wasextracted and was analyzed by TaqMan PCR for the presence of Lentiviralgenomes. FIG. 3A: The average copy number of lentiviral genomes from 4mice per group. FIG. 3B: The data for two individual portal vein (closedbars) or tail vein (open bars) LV-hFIX mice that expressed 159 nghFIX/ml serum and 67 ng hFIX/ml serum, respectively, at day 48.

[0013]FIGS. 4A-4I are photomicrographs showing expression of hFIX in theliver and spleen following LV-hFIX gene transfer. Livers and spleens ofLV-hFIX transduced mice were analyzed by immunohistochemistry for hFIXexpression 48 days following portal vein or tail vein delivery of 2×10⁸TU of LV-hFIX. Liver sections from PBS control (FIG. 4A), LV-hFIX portalvein (FIG. 4B) and LV-hFIX tail vein (FIG. 4C) mice are shown stained byanti-hFIX immunohistochemistry. Mouse liver sections were also stainedwith hematoxylin and eosin (H and E): PBS (FIG. 4D), portal vein (FIG.4E), tail vein (FIG. 4F). Sections from spleens of PBS control (FIG.4G), LV-hFIX portal vein (FIG. 4H) and LV-hFIX tail vein (FIG. 4I) miceare shown stained by anti-hFIX immunohistochemistry.

[0014] FIGS. 5A-F are photomicrographs showing expression of hFIX andPCNA in the liver following LV-hFIX gene transfer. Serial liver sectionsfrom PBS control (FIG. 5A, FIG. 5C, FIG. 5E) and LV-hFIX portal vein(FIG. 5B, FIG. 5D, FIG. 5F) mice for were stained for PCNA (FIG. 5A andFIG. 5B) or hFIX (FIG. 5C and FIG. 5D) expression 3 days followingportal vein delivery of 2×10⁸ TU of LV-hFIX. Sections were also stainedwith H and E (FIG. 5E and FIG. 5F). PCNA staining is denoted by theblack arrow and hFIX staining is denoted by the white arrow.

[0015] FIGS. 6A-H are graphs showing serum FIX expression followingvascular delivery of LV-hFIX. (FIG. 6A) Adult Swiss nude mice receivedeither 1.5×10⁸ TU (3 μg, ∘) or 1.5×10⁹ TU (30 μg, ) of LV-hFIX into theportal vein (four mice/group). hFIX levels were determined over time.Animals were killed after 122 days, and vector copy number in the liver(FIG. 6B) and spleen (FIG. 6C) were determined in a subset of animals.Serum levels of creatine phosphokinase (CPK), creatinine, alkalinephosphatase, albumin, and sGPT were determined at various time pointsimmediately after vector administration (FIG. 6D-FIG. 6H, respectively).

[0016]FIGS. 7A-7L are photomicrographs showing expression of hFIX in theliver after LV-hFIX gene transfer. Livers of three mice were analyzed byimmunohistochemistry for hFIX expression 122 days after portal veindelivery of LV-hFIX (1.5×10⁹ TU). Liver sections from PBS control (FIG.7A) and LV-hFIX portal vein mice (FIG. 7B-FIG. 7D) are shown stained byhFIX immunohistochemistry. Liver sections from PBS control (FIG. 7E) orLV-hFIX-treated mice (FIG. 7F-FIG. 7H) were stained using an anti-CD31(PECAM-1) antibody to identify the endothelial cells. Mouse liversections were also stained with H & E: PBS (FIG. 7I), LV-hFIX (FIG.7J-FIG. 7L). Scale bar, 10 μm.

[0017]FIG. 8A is a graph showing the kinetics of LV-luciferase (LV-luc)gene transfer. Adult Swiss nude mice received 2×10⁸ TU of LV-luc intothe portal vein, and the expression of luciferase was monitored in themice over time (three mice/group).

[0018]FIG. 8B is a typical image of a pair of LV-luc-treated and controlmice (as in FIG. 8A) 14 days after vector delivery, with the primaryorgans of LV transduction, the liver and spleen, indicated.

[0019] FIGS. 9A-L demonstrate that lentiviral transduction of the liverdoes not require cell proliferation.

[0020]FIGS. 9A-9B are liver sections of (FIG. 9A) control and (FIG. 9B)LV-GFP (4×10⁸ TU)-transduced mice (n=4) that were analyzed byfluorescence for GFP expression one week after portal vein delivery ofLU-GFP. FIG. 9C is a liver section from a third group of four mice thatreceived the LV-GFP after a partial hepatectomy.

[0021]FIG. 9D-FIG. 9F show liver sections from a subset of the mice inwhich BrdU was administered to the mice for seven days after vectoradministration to identify the cells that had proliferated.BrdU-positive cells are indicated by arrows. FIG. 9G-FIG. 9I showneighboring liver sections that were H & E stained. Scale bar, 10 μm.

[0022]FIG. 9J is a bar graph showing the percentage of GFP-positivecells. FIG. 9K is a bar graph showing liver lentiviral copy number: nopartial hepatectomy (empty bar), 1.9±0.8 copies/cell; with partialhepatectomy (filled bar), 2.9±1.3 copies/cell. FIG. 9L is a bar graphshowing the percentage of BrdU-positive liver cells.

[0023]FIG. 10 is a restriction map of the vectorpCCLsinL-F8.SMALL.ii.hF8pptpre.

DETAILED DESCRIPTION OF THE INVENTION

[0024] The invention is based on the discovery that replicationdeficient lentiviral vectors can be used to achieve stable geneticmodification of cells in vivo without vector-mediated toxicity and inthe absence of target cell proliferation. The examples disclosed hereindemonstrate that vascular and hepatic delivery of therapeutic doses of a3^(rd) generation lentiviral vector encoding human Factor IX (LV-hFIX)produce serum levels of hFIX with no vector mediated systemic toxicityof adult mice. Vascular delivery of the lentiviral vector results inpreferential transduction of the liver and spleen without anyconcomitant virus-mediated cytopathology. The invention thus providesvectors for transgene delivery as well as methods for gene therapy usingsuch vectors. The invention further provides promoters and enhancersuseful for optimizing tissue specific transgene delivery.

[0025] Definitions

[0026] All scientific and technical terms used in this application havemeanings commonly used in the art unless otherwise specified. As used inthis application, the following words or phrases have the meaningsspecified.

[0027] As used herein, a “second generation” lentiviral vector systemrefers to a lentiviral packaging system that lacks functional accessorygenes, such as one from which the accessory genes, vif, vpr, vpu andnef, have been deleted or inactivated. See, e.g., Zufferey et al., 1997,Nat. Biotechnol. 15:871-875.

[0028] As used herein, a “third generation” lentiviral vector systemrefers to a lentiviral packaging system that has the characteristics ofa second generation vector system, and that further lacks a functionaltat gene, such as one from which the tat gene has been deleted orinactivated. Typically, the gene encoding rev is provided on a separateexpression construct. See, e.g., Dull et al., 1998, J. Virol.72(11):8463-8471.

[0029] As used herein, “packaging system” refers to a set of viralconstructs comprising genes that encode viral proteins involved inpackaging a recombinant virus. Typically, the constructs of thepackaging system will ultimately be incorporated into a packaging cell.

[0030] As used herein, a “retroviral transfer vector” or “lentiviraltransfer vector” means an expression vector that comprises a nucleotidesequence that encodes a transgene and that further comprises nucleotidesequences necessary for packaging of the vector.

[0031] As used herein, “significant toxicity” means a level of toxicitythat contraindicates clinical use as determined by an art-acceptedmeasure of toxicity. Examples of art-accepted measures of toxicityinclude, but are not limited to, elevated serum levels of an enzyme orother substance associated with liver toxicity, such as sGPT,creatinine, alkaline phosphatase and alanine aminotransferase (ALT). Inone embodiment, elevated serum levels means higher than the upper limitof the normal range.

[0032] As used herein, “subject” refers to the recipient of the therapyto be practiced according to the invention. The subject can be anyanimal, including a vertebrate, but will preferably be a mammal. If amammal, the subject will preferably be a human, but may also be adomestic livestock, laboratory subject or pet animal.

[0033] As used herein, “transgene” means a polynucleotide that can beexpressed, via recombinant techniques, in a non-native environment orheterologous cell under appropriate conditions. The transgene may bederived from the same type of cell in which it is to be expressed, butintroduced from an exogenous source, modified as compared to acorresponding native form and/or expressed from a non-native site, or itmay be derived from a heterologous cell. “Transgene” is synonymous with“exogenous gene”, “foreign gene” and “heterologous gene”.

[0034] As used herein, a “therapeutic” gene means one that, whenexpressed, confers a beneficial effect on the cell or tissue in which itis present, or on a mammal in which the gene is expressed. Examples ofbeneficial effects include amelioration of a sign or symptom of acondition or disease, prevention or inhibition of a condition ordisease, or conferral of a desired characteristic. Therapeutic genesinclude genes that correct a genetic deficiency in a cell or mammal.

[0035] As used herein, a “promoter” means a nucleic acid sequencecapable of directing transcription.

[0036] As used herein, a “therapeutically acceptable amount” of asubstance means a sufficient quantity of the substance that anamelioration of adverse symptoms or protection against adverse symptomscan be detected in a subject treated with the substance.

[0037] As used herein, “expression control sequence” means a nucleicacid sequence that directs transcription of a nucleic acid. Anexpression control sequence can be a promoter, such as a constitutive oran inducible promoter, or an enhancer. The expression control sequenceis operably linked to the nucleic acid sequence to be transcribed.

[0038] As used herein, “nucleotide sequence”, “nucleic acid” or“polynucleotide” refers to a deoxyribonucleotide or ribonucleotidepolymer in either single- or double-stranded form, and unless otherwiselimited, encompasses known analogs of natural nucleotides that hybridizeto nucleic acids in a manner similar to naturally-occurring nucleotides.

[0039] As used herein, “pharmaceutically acceptable carrier” includesany material which, when combined with an active ingredient, allows theingredient to retain biological activity and is non-reactive with thesubject's immune system. Examples include, but are not limited to, anyof the standard pharmaceutical carriers such as a phosphate bufferedsaline solution, water, emulsions such as oil/water emulsion, andvarious types of wetting agents. Preferred diluents for aerosol orparenteral administration are phosphate buffered saline or normal (0.9%)saline.

[0040] Compositions comprising such carriers are formulated by wellknown conventional methods (see, for example, Remington's PharmaceuticalSciences, 18th edition, A. Gennaro, ed., Mack Publishing Co., Easton,Pa., 1990).

[0041] As used herein, “a” or “an” means at least one, unless clearlyindicated otherwise.

[0042] Lentiviral Vectors, Packaging Systems and Producer Cells

[0043] The invention provides lentiviral vectors, particles, packagingsystems and producer cells capable of producing a high titer recombinantlentivirus capable of selectively infecting human and other mammaliancells. In one embodiment, the recombinant lentivirus of the inventionhas a titer of 5×10⁵ infectious units/ml. Preferably, the recombinantretrovirus has a titer of 2×10⁶ infectious units/ml, and morepreferably, of 1×10⁷ infectious units/ml. Typically, titer is determinedby conventional infectivity assay on 293T, HeLa or HUH7 hepatoma cells.

[0044] Lentiviruses include members of the bovine lentivirus group,equine lentivirus group, feline lentivirus group, ovinecaprinelentivirus group and primate lentivirus group. The development oflentiviral vectors for gene therapy has been reviewed in Klimatcheva etal., 1999, Frontiers in Bioscience 4: 481-496. The design and use oflentiviral vectors suitable for gene therapy is described, for example,in U.S. Pat. No. 6,207,455, issued Mar. 27, 2001, and U.S. Pat. No.6,165,782, issued Dec. 26, 2000. Examples of lentiviruses include, butare not limited to, HIV-1, HIV-2, HIV-1 /HIV-2 pseudotype, HIV-1 /SIV,FIV, caprine arthritis encephalitis virus (CAEV), equine infectiousanemia virus and bovine immunodeficiency virus. HIV-1 is preferred.

[0045] Also provided is a lentiviral vector that contains a transgene,as well as a pharmaceutical composition comprising the lentiviral vectorand, optionally, a pharmaceutically acceptable carrier. The transgene istypically a therapeutic gene. An example of a therapeutic transgenedirected at treatment of hemophilia is one that encodes a blood clottingfactor, such as a polynucleotide encoding factor VIII or apolynucleotide encoding factor IX. In other examples, the therapeuticgene may be directed at cancer, infectious disease, a genetic deficiencyor other condition. Additional examples of therapeutic genes are thosethat encode cytokines, including interleukins and colony stimulatingfactors. Those skilled in the art will appreciate a variety oftransgenes that are suitable for use with the invention.

[0046] In one embodiment, the recombinant lentivirus can be used totransduce cells of a subject without resulting in significant toxicityor immunogenicity in the subject, and, following transduction, thetransgene is expressed. Upon transduction of the cells of a subject, thetherapeutic protein is expressed in a therapeutically acceptable amount.In some embodiments, the cells to be transduced are non-dividing cells,such as neuronal, muscle, liver, skin, heart, lung and bone marrowcells. In a preferred embodiment, the cells of the subject are livercells. Typically, the transgene is operatively linked to a promoter orother expression control sequence. An inducible promoter can be used forcontrolled expression of the transgene.

[0047] Expression Control Sequences

[0048] In a preferred embodiment, expression of the transgene is underthe control of a tissue specific promoter and/or enhancer. Preferably,the promoter or other expression control sequence selectively enhancesexpression of the transgene in liver cells. Examples of liver specificpromoters include, but are not limited to, the mouse thyretin promoter(mTTR), the endogenous human factor VIII promoter (F8), humanalpha-1-antitrypsin promoter (hAAT), human albumin minimal promoter, andmouse albumin promoter. The mTTR promoter is preferred. The mTTRpromoter is described in R. H. Costa et al., 1986, Mol. Cell. Biol.6:4697. The F8 promoter is described in Figueiredo and Brownlee, 1995,J. Biol. Chem. 270:11828-11838.

[0049] Expression levels can be further enhanced to achieve therapeuticefficacy using one or more enhancers. One or more enhancers can beprovided either alone or together with one or more promoter elements.Typically, the expression control sequence comprises a plurality ofenhancer elements and a tissue specific promoter. A preferred enhancercomprises one or more copies of the α-1-microglobulin/bikunin enhancer(Rouet et al., 1992, J. Biol. Chem. 267:20765-20773; Rouet et al., 1995,Nucleic Acids Res. 23:395-404; Rouet et al., 1998, Biochem. J.334:577-584; Ill et al., 1997, Blood Coagulation Fibrinolysis8:S23-S30). Also preferred are enhancers derived from liver specifictranscription factor binding sites, such as EBP, DBP, HNF1, HNF3, HNF4,HNF6, with Enh1, comprising HNF1 (sense)-HNF3 (sense)-HNF4(antisense)-HNF1 (antisense)-HNF6 (sense)-EBP (antisense)-HNF4(antisense), being most preferred (see Example 2 below).

[0050] As disclosed in Example 4 below, two copies ofα-1-microglobulin/bikunin enhancer, also referred to as Enhancer L, incombination with a ˜300 nucleotide fragment of the human Factor VIIIendogenous promoter (Figueiredo and Brownlee, 1995, J. Biol. Chem.270:11828-11838), has been shown to provide sustained expression (>14days) in the HemoA null mouse in vivo. As described in Example 3 below,the combination of Enh1 and the mTTR promoter enhances the activity overthe mTTR promoter alone by about two fold.

[0051] Packaging Systems

[0052] The invention is applicable to a variety of retroviral systems,and those skilled in the art will appreciate the common elements sharedacross differing groups of retroviruses. The description herein useslentiviral systems as a representative example. All retroviruses,however, share the features of enveloped virions with surfaceprojections and containing one molecule of linear, positive-sense singlestranded RNA, a genome consisting of a dimer, and the common proteinsgag, pol and env.

[0053] Lentiviruses share several structural virion proteins in common,including the envelope glycoproteins SU (gp120) and TM (gp41), which areencoded by env, and CA (p24), MA (p17) and NC (p7-11), which are encodedby the gag gene. HIV-1 and HIV-2 contain accessory and other proteinsinvolved in regulation of synthesis and processing virus RNA and otherreplicative functions. The accessory proteins, encoded by the vif, vpr,vpu/vpx, and nef genes, can be omitted (or inactivated) from therecombinant system. In addition, tat and rev can be omitted orinactivated.

[0054] First generation lentiviral vector systems provide separatepackaging constructs for gag/pol and env, and typically employ aheterologous envelope protein for safety reasons. In second generationlentiviral vector systems, the accessory genes, vif, vpr, vpu and nef,are deleted or inactivated. Third generation lentiviral vector systemsare those from which the tat gene has been deleted or otherwiseinactivated (e.g., via mutation).

[0055] Compensation for the regulation of transcription normallyprovided by tat can be provided by the use of a strong constitutivepromoter, such as the human cytomegalovirus immediate early (HCMV-IE)enhancer/promoter. Other promoters/enhancers can be selected based onstrength of constitutive promoter activity, specificity for targettissue (e.g., liver-specific promoter), or other factors relating todesired control over expression, as is understood in the art. Forexample, in some embodiments, it is desirable to employ an induciblepromoter such as tet to achieve controlled expression. The gene encodingrev is preferably provided on a separate expression construct, such thata typical third generation lentiviral vector system will involve fourplasmids: one each for gagpol, rev, env and the transfer vector.Regardless of the generation of packaging system employed, gag and polcan be provided on a single construct or on separate constructs.

[0056] Accordingly, the invention provides a retroviral packaging systemthat comprises at least two vectors: a first packaging vector comprisinga gag, a pol, or gag and pol genes operably linked to an expressioncontrol sequence; and a second packaging vector comprising aheterologous env gene operably linked to an expression control sequence.For production of recombinant lentiviral vectors of the invention, thesystem further comprises a lentiviral transfer vector that comprises agene that encodes a blood clotting factor operably linked to anexpression control sequence. Typically, the set of lentiviral vectorslacks a functional tat gene.

[0057] Preferably, the heterologous env gene comprises a VSV-G orbaculoviral gp64 env gene, although those skilled in the art willappreciate other suitable env genes. Pseudotyping with a baculoviral envgene, for example, can reduce toxicity and avoid an overly broad tropismthat can lead to an undesired immune response directed against therecombinant viral vector. Representative gp64 genes and methods forpreparing them are described in Monsma & Blissard, 1995, J. Virol.69(4):2583-95; Blissard & Rohrmann, 1989, Virology 170(2):537-55; andBlissard & Monsma, U.S. Pat. No. 5,750,383. The gp64 or otherbaculoviral env gene can be derived from Autographa californicanucleopolyhedrovirus (AcMNPV), Anagrapha falcifera nuclear polyhedrosisvirus, Bombyx mori nuclear polyhedrosis virus, Choristoneura fumiferananucleopolyhedrovirus, Orgyia pseudotsugata single capsid nuclearpolyhedrosis virus, Epiphyas postvittana nucleopolyhedrovirus,Hyphantria cunea nucleopolyhedrovirus, Galleria mellonella nuclearpolyhedrosis virus, Dhori virus, Thogoto virus Antheraea pernyinucleopolyhedrovirus or Batken virus. Preferably, the gp64 env gene isan AcMNPV gp64 env gene.

[0058] In a preferred embodiment, the retroviral elements are derivedfrom a lentivirus, such as HIV. Preferably, the vectors lack afunctional tat gene and/or functional accessory genes (vif, vpr, vpu,vpx, nef). In another preferred embodiment, the system further comprisesan additional vector that comprises a rev gene.

[0059] The vectors of the packaging system are expression constructsthat include the gene(s) encoding retroviral packaging elements, whereineach gene is operably linked to an expression control sequence. In oneembodiment, the vector is a plasmid. Other vectors, however, are knownin the art and include, for example, viral vectors.

[0060] Typically, the vectors are included in a packaging cell. Thevectors are introduced via transfection, transduction or infection intothe packaging cell line. Methods for transfection, transduction orinfection are well known by those of skill in the art. A transfer vectorcan be introduced into the packaging cell line, via transfection,transduction or infection, to create a producer cell. The producer cellproduces viral particles that contain the transgene. The recombinantvirus is recovered from the culture media and titrated by standardmethods used by those of skill in the art.

[0061] The packaging constructs can be introduced into human cell linesby calcium phosphate transfection, lipofection or electroporation,generally together with a dominant selectable marker, such as neo, DHFR,Gln synthetase or ADA, followed by selection in the presence of theappropriate drug and isolation of clones. A selectable marker gene canbe linked physically to the packaging genes in the construct.

[0062] Stable cell lines, wherein the packaging functions are configuredto be expressed by a suitable packaging cell, are known. For example,see U.S. Pat. No. 5,686,279; and Ory et al., Proc. Natl. Acad. Sci.(1996) 93:11400-11406, which describe packaging cells. Furtherdescription of stable cell line production can be found in Dull et al.,1998, J. Virology 72(11):8463-8471; and in Zufferey et al., 1998, J.Virology 72(12):9873-9880

[0063] Zufferey et al., 1997, Nature Biotechnology 15:871-875, teach alentiviral packaging plasmid wherein sequences 3′ of pol including theHIV-1 env gene are deleted. The construct contains tat and rev sequencesand the 3′ LTR is replaced with poly A sequences. The 5′ LTR and psisequences are replaced by another promoter, such as one that isinducible. For example, a CMV promoter or derivative thereof can beused.

[0064] The packaging vectors of interest may contain additional changesto the packaging functions to enhance lentiviral protein expression andto enhance safety. For example, all of the HIV sequences upstream of gagcan be removed. Also, sequences downstream of env can be removed.Moreover, steps can be taken to modify the vector to enhance thesplicing and translation of the RNA.

[0065] Optionally, a conditional packaging system is used, such as thatdescribed by Dull et al., 1998, J. Virology 72(11):8463-8471. Alsopreferred is the use of a self-inactivating vector (SIN), which improvesthe biosafety of the vector by deletion of the HIV-1 long terminalrepeat (LTR) as described, for example, by Zufferey et al., 1998, J.Virology 72(12):9873-9880. Inducible vectors can also be used, such asthrough a tet-inducible LTR.

[0066] The techniques used to construct vectors, and to transfect and toinfect cells, are practiced widely in the art. Practitioners arefamiliar with standard resource materials that describe specificconditions and procedures. Construction of the vectors of the inventionemploys standard ligation and restriction techniques that are wellunderstood in the art (see Maniatis et al., in Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, N.Y., 1982). Isolatedplasmids, DNA sequences or synthesized oligonucleotides are cleaved,tailored and religated in the form desired.

[0067] Conventional methods can be used to propagate the viruses used inthe invention (see, e.g., Burleson, et al., 1992, Virology: A LaboratoryManual, Academic Press, Inc., San Diego, Calif.; and Mahy, ed., 1985,Virology: A Practical Approach, IRL Press, Oxford, UK). Conventionalconditions for propagating viruses are suitable for allowing expressionof a baculoviral envelope protein on the surface of a retrovirusparticle used in the invention.

[0068] Methods for the large-scale production of safe and efficientretrovirus packaging lines for use in immunotherapy protocols isdescribed in Farson et al., 1999, J. Gene Medicine 1:195-209. Additionalguidance on the production and use of lentiviral vectors is provided inU.S. Pat. No. 6,165,782, issued Dec. 26, 2000, and in PCT ApplicationNo. US 00/11097, published Nov. 29, 2000. Transduction efficiency can beenhanced and toxicity minimized or eliminated through the selection ofelements for the vector construct as well as through vectorpurification.

[0069] Producer cells of the invention comprise packaging constructs asdescribed above, as well as an expression construct that comprises atransgene of interest operably linked to an expression control sequence.This expression construct is also referred to as a transfer vector.

[0070] Preferred is the use of recombinant lentiviral vectors that arecapable of high infectivity (e.g., more than 20% of target cellsexpressing transgene, preferably more than 25% of target cellsexpressing, or an infectivity of at least about 5×10⁷ TU/μg p24 Gagantigen) of quiescent as well as proliferating cells. Also preferred isthe use of a purification protocol sufficient to produce a viral stockthat is substantially free of non-infectious contaminants. In apreferred embodiment, the vectors are centrifuged at low speed,filtered, and then concentrated by high speed centrifugation, such as atabout 19,500 rpm.

[0071] Transgenes

[0072] The transgene can be any nucleic acid of interest that can betranscribed. Generally the transgene encodes a polypeptide. Preferablythe polypeptide has some therapeutic benefit. The polypeptide maysupplement deficient or nonexistent expression of an endogenous proteinin a host cell, or the polypeptide can confer new properties on the hostcell, such as a chimeric signaling receptor (see U.S. Pat. No.5,359,046). The artisan can determine the appropriateness of aparticular transgene by using techniques known in the art. For example,the artisan would know whether a foreign gene is of a suitable size forencapsidation and whether the foreign gene product is expressedproperly.

[0073] It may be desirable to modulate the expression of agene-regulating molecule in a cell by the introduction of a molecule bythe method of the invention. The term “modulate” refers to thesuppression of expression of a gene when it is over-expressed, or toaugmentation of expression when it is under-expressed. Where a cellproliferative disorder is associated with the expression of a gene,nucleic acid sequences that interfere with the expression of a gene atthe translational level can be used. The approach can utilize, forexample, antisense nucleic acid, ribozymes or triplex agents to blocktranscription or translation of a specific mRNA, either by masking thatmRNA with an antisense nucleic acid or triplex agent, or by cleavingsame with a ribozyme.

[0074] Antisense nucleic acids are DNA or RNA molecules that arecomplementary to at least a portion of a specific mRNA molecule(Weintraub, 1990, Sci. Am. 262:40). In the cell, the antisense nucleicacids hybridize to the corresponding mRNA, forming a double-strandedmolecule. The antisense nucleic acids interfere with the translation ofthe mRNA, as the cell will not translate a mRNA that is double-stranded.Antisense oligomers of about 15 nucleotides or more are preferredbecause such are synthesized easily and are less likely to causeproblems than larger molecules when introduced into the target cell. Theuse of antisense methods to inhibit the in titro translation of genes iswell known in the art (Marcus-Sakura, 1988, Anal. Biochem. 172:289).

[0075] The antisense nucleic acid can be used to block expression of amutant protein or a dominantly active gene product, such as amyloidprecursor protein that accumulates in Alzheimer's disease. Such methodsare also useful for the treatment of Huntington's disease, hereditaryParkinsonism and other diseases. Antisense nucleic acids are also usefulfor the inhibition of expression of proteins associated with toxicity.

[0076] Use of an oligonucleotide to stall transcription can be by themechanism known as the triplex strategy since the oligomer winds arounddouble-helical DNA, forming a three-strand helix. Therefore, the triplexcompounds can be designed to recognize a unique site on a chosen gene(Maher et al., 1991, Antisense Res and Dev. 1(3):227; Helene, 1991,Anticancer Drug Dis. 6(6):569).

[0077] Ribozymes are RNA molecules possessing the ability tospecifically cleave other single-stranded RNA in a manner analogous toDNA restriction endonucleases. Through the modification of nucleotidesequences which encode those RNA's, it is possible to engineer moleculesthat recognize and cleave specific nucleotide sequences in an RNAmolecule (Cech, 1988, J. Amer. Med Assn. 260:3030). A major advantage ofthat approach is only mRNAs with particular sequences are inactivated.

[0078] It may be desirable to transfer a nucleic acid encoding abiological response modifier. Included in that category areimmunopotentiating agents including nucleic acids encoding a number ofthe cytokines classified as interleukins, for example, interleukins 1through 12. Also included in that category, although not necessarilyworking via the same mechanism, are interferons, and in particular,gamma interferon (γ-IFN), tumor necrosis factor (TNF) andgranulocyte-macrophage colony stimulating factor (GM-CSF). It may bedesirable to deliver such nucleic acids to bone marrow cells ormacrophages to treat inborn enzymatic deficiencies or immune defects.Nucleic acids encoding growth factors, toxic peptides, ligands,receptors or other physiologically important proteins also can beintroduced into specific non-dividing cells.

[0079] Thus, the recombinant lentivirus of the invention can be used totreat an HIV-infected cell (e.g., T-cell or macrophage) with an anti-HIVmolecule. In addition, respiratory epithelium, for example, can beinfected with a recombinant lentivirus of the invention having a genefor cystic fibrosis transmembrane conductance regulator (CFTR) fortreatment of cystic fibrosis.

[0080] The method of the invention may also be useful for neuronal,glial, fibroblast or mesenchymal cell transplantation, or grafting,which involves transplantation of cells infected with the recombinantlentivirus of the invention ex vivo, or infection in vivo into thecentral nervous system or into the ventricular cavities or subdurallyonto the surface of a host brain. Such methods for grafting are known tothose skilled in the art and are described in Neural Grafting in theMammalian CNS, Bjorklund & Stenevi, eds. (1985).

[0081] For diseases due to deficiency of a protein product, genetransfer could introduce a normal gene into the affected tissues forreplacement therapy, as well as to create animal models for the diseaseusing antisense mutations. Preferably, the transgene encodes a bloodclotting factor, such as Factor VIII or IX, and the target cell is aliver cell.

[0082] Methods

[0083] The invention provides a method of producing a recombinantlentivirus. The method comprises transforming a host cell with a firstnucleotide sequence comprising a gag, a pol, or gag and pol genes; and asecond nucleotide sequence comprising a heterologous env gene.Preferably, the env gene comprises a VSV-G or gp64 env gene. In apreferred embodiment, the lentiviral elements are derived from HIV, suchas HIV-1. Preferably, the vectors lack a functional tat gene and/orfunctional accessory genes (vif, vpr, vpu, vpx, nef). In anotherpreferred embodiment, the method further comprises transforming the hostcell with an additional nucleotide sequence that comprises a rev gene.The method further comprises transforming the host cell with a transfervector comprising a transgene operably linked to an expression controlsequence. Preferably, the transgene is a therapeutic transgene, such asa transgene that encodes a blood clotting factor (e.g., Factor VIII orFactor IX). Preferred expression control sequences include tissuespecific promoters and/or enhancers, such as one of the liver specificpromoters or enhancers disclosed herein. The host cell can be culturedunder conditions suitable for viral production, and recombinant viruscan be recovered from the culture medium.

[0084] The invention additionally provides methods for delivering atransgene to a cell, in vivo, in vitro or ex vivo. Also provided aremethods of treating a subject and of delivering a therapeutic transgeneto cells of a subject. In one embodiment, the subject is treated forhemophilia and the transgene comprises a blood clotting factor. Thetransgene can be delivered to dividing or to quiescent cells in asubject, such as liver cells. The method comprises transducing a cellwith a recombinant lentiviral vector that contains a transgene.Preferably, the transgene is a therapeutic transgene. In a typicalembodiment, significant toxicity is not caused in the subject. Toxicitycan be minimized or eliminated by use of a vector of the invention, suchas those described herein and having an infectivity of at least about5×10⁷ TU/μg p24 Gag antigen.

[0085] Vectors of the invention can be administered to a subjectparenterally, preferably intravascularly (including intravenously). Whenadministered parenterally, it is preferred that the vectors be given ina pharmaceutical vehicle suitable for injection such as a sterileaqueous solution or dispersion. Following administration, the subject ismonitored to detect changes in gene expression. Dose and duration oftreatment is determined individually depending on the condition ordisease to be treated. A wide variety of conditions or diseases can betreated based on the gene expression produced by administration of thegene of interest in the vector of the present invention. The dosage ofvector delivered using the method of the invention will vary dependingon the desired response by the host and the vector used. Generally, itis expected that up to 100-200 μg of DNA or RNA can be administered in asingle dosage, although a range of 0.5 mg/kg body weight to 50 mg/kgbody weight will be suitable for most applications.

EXAMPLES

[0086] The following examples are presented to illustrate the presentinvention and to assist one of ordinary skill in making and using thesame. The examples are not intended in any way to otherwise limit thescope of the invention. Various references are indicated by numberspresented in superscript form. The citations for these references areprovided in a list at the conclusion of the Examples.

Example 1

[0087] Production of Human Factor IX without Toxicity in Mice afterVascular Delivery of a Lentiviral Vector

[0088] Replication deficient lentiviral vectors have been shown toenable the stable genetic modification of multiple cell types in vivo.This example demonstrates that vascular and hepatic delivery oftherapeutic doses of a 3^(rd) generation HIV derived lentiviral vectorencoding human Factor IX (LV-hFIX) produce therapeutic serum levels ofhFIX protein with no vector mediated local or systemic toxicity of adultmice. Vascular delivery of the lentiviral vector resulted in thepreferential transduction of the liver and spleen without anyconcomitant virus-mediated cytopathology. Cell proliferation was notrequired for hepatocyte transduction with either a LV encoding hFIX or aLV encoding GFP. Portal vein administration produced the highest serumlevels of hFIX and demonstrated proportionally higher levels of genetransfer to the liver with up to 35% of cells, hepatocytes, endothelialcells and Kupffer cells, expressing hFIX. Serum hFIX levels reached 4%of normal levels following vascular LV-mediated hFIX gene transfer andremained stable for months following vector administration.

[0089] Introduction

[0090] Replication deficient murine MLV based retroviruses have beenemployed for years in the ex vivo genetic modification of cells as wellas in a limited number of in vivo gene therapy protocols. These vectors,while suitable for many applications, have the significant disadvantageof requiring DNA replication and the breakdown of the nuclear membranethat accompanies cell division to stably transduce target cells.Replication deficient lentivirus vectors were developed to overcome thislimitations¹. Unlike MLV based retroviral vectors, lentiviral vectorsefficiently transduce both growth arrested and proliferating cells invitro and in vivo¹⁻⁵. Typical 3^(rd) generation lentiviral transfervectors retain less than 8% of the wild type HIV (wtHIV) sequences andhave been deleted of the HIV gag, pol and env genes as well as the HIVaccessory genes; vpr, vpu, vif and nef^(4,6). Further deletions in the3′ LTR of the vector have been made to create self-inactivating vectorsthat do not mobilize following integration into the target cell, evenupon subsequent infection with wtHIV^(7,8). The development oflentiviral vector producer cell lines now facilitate the manufacture oflarge quantities of the vector⁹.

[0091] Lentiviral vectors have been shown to stably transduce liver,muscle, skin, retina and brain in mice, rats or dogs following vascular,intramuscular, intradermal, intraoccular and intracranial administrationrespectively^(1-5,10-12). The cells in several of these tissues aretypically in a quiescent state. In contrast with these reports, it hasbeen recently suggested that liver directed delivery of lentiviralvectors does not result in transduction of hepatocytes in the absence ofcell division or DNA replication^(13,14).

[0092] The impact of the route of administration of a 3^(rd) generationlentiviral vector on vector biodistribution, hFIX expression and vectorsafety in adult mice was examined. The vascular administration of 2×10⁸transducing units (TU) (4 μg of p24) of a 3^(rd) generation VSV-Gpseudotyped lentiviral vector encoding human Factor IX (hFIX) in adultSwiss nude mice produced significant stable serum levels of hFIX withoutany evidence of local or systemic toxicity. Quantitative PCR analysis of10 tissues indicated that over 80% of vector genomes were found in theliver following intraportal administration of vector.Immunohistochemistry indicated that up to 35% of the hepatocytes andsinusoidal cells expressed the hFIX protein. Peripheral vascular (tailvein) administration of the vector resulted in lower serum levels ofhFIX, lower genome copies in the liver, and lower levels of hFIXpositive liver cells. There was however an increase in the transductionof the spleen with tail vein administration. These results demonstratethat vascular administration of lentiviral vectors is an efficient andsafe means of introducing the FIX gene into the liver.

[0093] Results

[0094] Safe and Efficient Vascular Deliver of Lentiviral Vector

[0095] The liver is the natural site for the expression of numeroussecreted proteins including hFIX¹⁷. Examined here was the efficacy ofhFIX gene delivery using a 3^(rd) generation VSV-G pseudotypedlentiviral vector encoding hFIX (LV-hFIX) delivered by multiple routesto access the liver. Four μg p24 (2×10⁸ TU) of LV-hFIX was administeredto adult Swiss nude mice by intraportal vein, intra-tail vein, splenicor direct liver injection (FIG. 1A). hFIX was detected in the serum assoon as 3 days following gene transfer in all mice. In mice thatreceived LV-hFIX by the intraportal route, hFIX levels increased overseveral weeks following gene transfer and serum hFIX reached peak levelsof 170 ng hFIX/ml (FIG. 1A, closed squares). Serum hFIX levels werelower in mice that received LV-hFIX by the tail vein (FIG. 1A, opensquares), splenic (FIG. 1A, closed triangles) or direct liver (FIG. 1A,open triangles) routes reaching maximal levels of 121 ng hFIX/ml.

[0096] To ascertain whether the LV-hFIX delivered to the portal vein ortail vein induced any systemic cytopathology, serum samples were testedfor elevation of serum markers of toxicity. At all time points examined,1, 3, 7 and 14 days following vector administration, sGPT, creatinineand alkaline phosphatase levels were within the normal range and wereindistiguishable from the PBS control animals (FIGS. 1B-D). Thisanalysis was expanded to include other routes of vector administrationand other markers of systemic toxicity. LV-hFIX delivered by eitherintraportal vein, intra-tail vein, splenic or direct liver routes didnot induce any significant increase in serum sGPT (FIG. 1E), creatinine(FIG. 1F), alkaline phosphate (FIG. 1G) or albumin (FIG. 1H) levels. Forall test groups, levels of these markers of liver, kidney and cardiactoxicity remained in the normal range at both early and late time pointsfollowing gene transfer and were indistinguishable from PBS injectedcontrol animals. Similar results were obtained with C57Bl/6 mice. Theseresults indicate that lentiviral vector gene transfer did not induce orrequire vector-induced cytopathology.

[0097] In a second study, Swiss nude mice were dosed with either 1.5×10⁸transducing units (TU) (3 μg) or 1.5×10⁹ TU (30 μg) of LV-hFIX into theportal vein, and sera were collected at various time points (FIG. 6A).TaqMan PCR analysis of livers of these mice 48 days after vectoradministration indicated that increasing the vector dose 10-foldproduced only a modest increase in vector genomes in the liver (FIG.6B). The spleens of the LV-hFIX mice were positive for vector genomes,although the vector genome levels were significantly lower than in theliver (FIG. 6C). No significant changes in serum creatine phosphokinase(CPK), creatinine, alkaline phosphatase, or albumin levels were observedat either dose or at any time point after vector administration (FIGS.6D-G). A slight increase in serum glutamic-pyruvic transaminase (sGPT)levels was observed one day after vector administration in the high-dosegroup (FIG. 6H). However, sGPT levels returned to normal at three daysafter vector administration. Even on day 1, the peak levels of sGPT inthe 30 μg LV-FIX group were within the normal range for mice (<50 IU).Mice that received LV-hFIX by the intraportal route showed average serumhFIX levels of 140 ng/ml (n=4). Serum hFIX levels were lower in micethat received LV-hFIX by the tail vein route, with average serum levelsof 70 ng hFIX/ml (n=4). Serum markers were all in the normal range forboth groups of mice.

[0098] To determine if LV-hFIX gene delivery was mouse strain dependent,2×10⁸ TU of LV-hFIX was administered to the tail vein or portal vein ofSwiss nude and C57BL/6 mice and the serum hFIX levels measured. Thekinetics of hFIX expression in C57Bl6 mice following portal veinadministration of LV-hFIX (FIG. 2B) was faster than that observed in theSwiss nudes (FIG. 2A), however, serum hFIX protein levels dropped by 14days. The levels of hFIX in the C57BL/6 mice that received the vectorvia the tail vein were lower and were also diminished over time. As theC57Bl6 mice are immunocompetent, one explanation for the disappearanceof hFIX protein might be the induction of an anti-human hFIX humoralimmune response in the mice. Indeed, high titer anti-hFIX antibodieswere detected in the serum of the LV-hFIX transduced C57BL/6 mice andthe kinetics of the appearance of these antibodies corresponded with theloss of serum hFIX expression (FIG. 2C). Despite the induction of ananti-hFIX response, there were no apparent changes in serum toxicitymarker levels in these mice.

[0099] To determine the target organs of the LV-hFIX, DNA was isolatedfrom various tissues from LV-hFIX transduced mice and the LV copynumber/1000 cells determined by quantitative PCR (FIG. 3A). In mice thatreceived the LV-hFIX by the portal vein route the liver was thepredominant tissue transduced (FIG. 3A, black bars). The livers of thesemice demonstrated approximately 350 vector genomes/1000 cells. Thespleen demonstrated approximately ½ of number of vector genomes observedin the liver with 150 vector genomes/1000 cells. A very minor fractionof the vector genomes were present in the lung. All other tissuessampled, kidney, heart, brain, pancreas, duodenum, testis, mesentericlymph nodes, possessed less than 0.2% of the total vector genomesdetected with most of the samples being negative. A similar analysis wasperformed with mice that received the LV-hFIX by the tail vein route.Again the liver and spleen were the primary tissues that were transducedby the vector (FIG. 3A, white bars). Both of these tissues possessedapproximately 150 vector genomes/1000 cells. All other tissuesdemonstrated minimal number of vector genomes.

[0100] The number of lentiviral vector genomes in the liver appeared tocorrelate with the serum hFIX levels. In FIG. 3B is shown thequantification of vector genomes for two individual mice that receivedvector by either the portal vein (FIG. 3B, black bars) or tail vein(FIG. 3B, white bars). In the portal vein LV-hFIX mouse that expressed159 ng hFIX/ml, 1085 lentiviral vector genome copies/1000 liver cellswere detected. The tail vein LV-hFIX mice expressed approximately 2 foldlower levels of hFIX (67 ng hFIX/ml) and possessed approximately 2 foldlower lentiviral vector genome copies in the liver cells (465copies/1000 cells). By contrast these two mice demonstrated similarlevels of vector genomes in their spleens. No other tissue in these miceexpressed significant number of lentiviral vector genomes. These datasuggested that the liver might be the primary site of hFIX expression.

[0101] To demonstrate hFIX protein expression in the livers of theLV-hFIX mice, hFIX immunohistochemistry was performed on livers fromanimals that received LV-hFIX by portal vein and tail vein 48 daysfollowing gene transfer. The staining procedure used here did not crossreact with the endogenous murine FIX protein as demonstrated in the PBScontrol mice, which did not exhibit significant HRP (brown) staining(FIG. 4A). In contrast, 25% to 35% of the liver cells in the portal veinLV-hFIX mice stained positive for hFIX (FIG. 4B, arrows). Staining waslocalized to both hepatocytes and liver sinusoidal cells. Thehepatocytic staining was patchy and moderate (FIG. 4B) while the liversinuses stained strongly positive for hFIX. Liver sections were stainedwith Perl's Prussian blue stain for Kupffer cells or with anti-CD34 toindicate endothelial cells that make up the sinuses. Endothelial cellsin the liver sinuses stained strongly positive for hFIX. Some Kupffercells also stained positive for hFIX.

[0102] Mice that received LV-hFIX by tail vein administrationdemonstrated reduced hFIX staining in the liver relative to the portalvein treated animals with approximately 5% to 15% of the liver cellsstaining positive for hFIX. The intensity of hFIX staining was alsoreduced in both hepatocytes and liver sinuses (FIG. 4C, arrow).Hematoxylin and eosin staining of liver sections from the LV-hFIXtransduced animals showed no signs of vector mediated liver toxicity(FIGS. 4E and F) compared to PBS control mice (FIG. 4D), consistent withthe serum marker results.

[0103] The quantitative PCR analysis of vector genomes indicated thatthe spleen was an important site of transduction for the LV-hFIX by bothroutes of administration. Immunohistochemistry staining for hFIX wasperformed on spleens from animals that received LV-hFIX by portal veinand tail vein 48 days following gene transfer. As expected no hFIXstaining was observed in the spleens of the PBS control mice (FIG. 4G).In the tail vein LV-hFIX mice, however, approximately 10% of the cellsof the spleen stained positive for hFIX staining (FIG. 4I), andapproximately 10% of the splenocytes in the tail vein LV-hFIX micestained positive for hFIX (FIG. 4I). In both sets of animals, splenichFIX staining appeared to be predominantly in lymphocytic cells. AsSwiss nude mice are T cell deficient, these cells are likely to be Bcells.

[0104] This study was repeated by delivering 4×10⁸ TU of a LV-GFP vectorinto the tail vein of C57BL/6 mice. The presence of GFP expression inimmune cells (i.e., the T cells, B cells, and antigen-presenting cells)was determined by fluorescence-activated cell sorting (FACS) five daysafter vector administration. The results are shown in Table 1. Overall,2.6% of the splenocytes expressed GFP. The majority of these GFP+ cellswere MHC class II positive, with approximately half being B cells. SomeGFP expression was also observed in T cells. The transduction andexpression of the transgene in antigen-presenting cells in C57BL/6 micemay be responsible for the induction of the robust anti-hFIX humoralimmune response observed in these immunocompetent mice following LV-hFIXadministration. TABLE 1 Transgene expression in splenocytes* TreatmentGFP+ GFP+/CD3+ GFP+/B220+ GFP+/MHC II+ Control 0.32% 0.03% 0.13% 0.31%LV-GFP 2.61% 0.37% 1.32% 2.44%

[0105] In addition, Immunohistochemistry for hFIX was performed onlivers from animals that received LV-hFIX by portal vein 122 days aftergene transfer. The staining procedure used here did not cross-react withthe endogenous murine FIX protein, as demonstrated in the PBS controlliver, which did not exhibit any horseradish peroxidase (HRP)-positive(brown) staining (FIG. 7A). In contrast, ˜4% of the liver cells in theLV-hFIX-transduced mice stained positively for hFIX (FIGS. 7B-D,arrows). As demonstrated in the three representative sections of FIGS.7B-D, hFIX-positive cells were observed in most sections examined. Liversections were stained with anti-CD31 (PECAM-1) to mark the endothelialcells that line the liver sinuses. The pattern of staining of thesecells (FIGS. 7E-H) was very different from that seen with thehFIX-stained sections. Although the occasional endothelial cell (emptyarrow) stained positively for hFIX, staining was predominantly localizedto the hepatocytes (filled arrows), as indicated by the morphology ofthe hFIX-positive cells and the distinct pattern of staining observedwith the hFIX and CD31 immunohistochemical stains. A similar pattern ofgene expression was also observed following vascular delivery of aLV-GFP vector. Hematoxylin and eosin (H & E) staining of sections fromthe LV-hFIX-transduced mice indicated normal liver architecture and theabsence of inflammatory cell infiltrates in the control andgene-modified mice (FIGS. 7J-L), consistent with the normal serum markerlevels.

[0106] The kinetics of gene expression after vector administration wasexamined by injecting 2×10⁸ TU of a lentiviral vector encoding theluciferase gene (LV-Luc) into the portal vein of adult mice. At varioustime points following vector administration, luciferase gene expressionwas quantified by administering luciferin to the mice and imaging with aXenogen (Alameda, Calif.) IVIS camera system. As soon as three daysfollowing LV-Luc administration, luciferase gene expression could bedetected in the liver and spleen (FIG. 8A). Gene expression stabilizedafter two to three weeks and was stable over the course of theexperiment. A typical image of mice that received either the LV-Luc orno vector is shown two weeks after vector administration (FIG. 8B). Theluminescence observed in the hindlimbs was not consistently observedwith subsequent imaging and may either represent background signal or alow-level signal from transduced bone marrow in the leg.

[0107] It has been suggested that lentiviral vectors require DNAreplication to transduce hepatocytes¹⁴. To address this question, liversections taken from mice that received 2×10⁸ TU of LV-hFIX via theportal vein 3 days following gene transfer were stained for theexpression of PCNA. PCNA is a transcription factor that is upregulatedduring the G₁/S phase transition and through DNA replication. In the PBScontrol and LV-hFIX transduced mice, 0.3-0.5% of the hepatocytes stainedpositive for PCNA 3 days following portal vein delivery of Lentivirus(FIGS. 5A and B). In contrast, approximately 10-15% of liver cells inthe LV-hFIX treated mouse stained positive for hFIX (FIG. 5D). No hFIXexpression was observed in serial sections in the liver from the PBScontrol mouse (FIG. 5C). From this analysis, it appears that vasculardelivery of lentiviral vectors does not induce hepatocellularcytopathology or hepatic cell division (FIGS. 5E and F). Furthermore,the transduction of hepatocytes by lentiviral vector does not requirecell division.

[0108] To further assess whether hepatocyte cell division is requiredfor lentiviral vector-mediated gene transfer, a partial hepatectomy wasperformed on mice before portal vein administration of the LV-GFPvector. Liver sections from these mice were compared to those fromnonhepatectomized LV-GFP-transduced mice. Although no GFP expression wasobserved in control mice (FIG. 9A), it was readily detected in thelivers of nonhepatectomized mice that received LV-GFP (FIG. 9B). In micethat received a partial hepatectomy (FIG. 9C), the transductionefficiency was higher, and transduced cells were larger in size andbrighter than in the nonhepatectomized mice. Quantification of GFPexpression indicated that ˜10% of the hepatocytes expressed GFP in theLV-GFP mice while ˜40% of liver cells in the LV-GFP transduced mice thathad been hepatectomized expressed GFP (FIG. 9J). Approximately 2 vectorcopies/cell were detected in the control LVGFP livers, and thehepatectomized animals showed approximately 3 vector genomes/cell (FIG.9K). In a subset of the mice, 5-bromo-2′-deoxyuridine (BrdU) wasadministered for seven days after vector administration to identify thecells that had proliferated. Similar levels of BrdU-positive cells wereobserved in the nonhepatectomized LV-GFP and PBS control mice (<1%;FIGS. 9D, E, arrows). As expected, the number of BrdU-positive cells wasincreased in the hepatectomized LV-GFP mice (>40%) (FIG. 9F). Analysisof H & E-stained liver sections from these mice indicated that there wasno vector-mediated cytopathology or inflammatory infiltrates (FIG. 9H),and the transduced liver sections were comparable to the normal controls(FIG. 9G).

[0109] Discussion

[0110] Lentiviral vectors have been shown to transduce a variety of celltypes both in vitro and in vivo, including cell types that are normallyquiescent. As shown here, vascular administration of a VSV-G pseudotyped3^(rd) generation lentiviral vector encoding human factor IX resulted inthe significant transduction of hepatocytes and liver sinus endothelialcells and produced therapeutic levels of hFIX in the serum of mice. Upto 12-35% of the liver cells were observed to express hFIX, depending onroute of administration, and maximal hFIX serum levels were 200 nghFIX/ml following portal vein administration of 1.5×10⁹ TU of LV-hFIX.

[0111] Serum hFIX levels reached 4% of normal levels following vascularlentiviral-mediated hFIX gene transfer, and remained stable for monthsfollowing vector administration. Human FIX levels as low as 1-2% ofnormal are considered sufficient to achieve a therapeutic effect. Alevel of 200 ng/ml would put a severe hemophiliac into the moderaterange for FIX.

[0112] No evidence of concomitant local or systemic cytopathology wasobserved in the treated mice. No induction of hepatocytic DNAreplication was detected following vector administration, and there wasno apparent correlation between the cycling hepatocytes and those thatexpressed hFIX.

[0113] Park et al. reported that doses of 8-10×10⁸ TU of LV-LacZ vectorwere required to achieve significant transduction of the liver followingportal vein administration of vector¹⁴. In addition, Park et al.reported that lentivirus vector at these doses mediated significanthepatocellular toxicity and that 74% (20/27) of mice died followingadministration of 8-10×10⁸ TU of LV-LacZ¹⁴. This level of toxicity hasnot been observed in the studies described herein or in studiesemploying LV-hFIX doses up to 4×10⁸ TU and LV-LacZ doses up to 8×10⁸ TU.Park et al. also reported that over 100 μg of p24 per dose of 2^(nd)generation LV-hFIX was required to achieve therapeutic serum levels ofhFIX¹³. These amounts of virus (p24 equivalents) were 10- to 25-foldhigher than those required in the current study.

[0114] Park et al. also reported minimal lentivirus vector transductionof the liver in the absence of hepatocyte replication. The transductionefficiency was improved when vector was administered following a partialhepatectomy of the animals. In the current study, using a similar dose(p24 equivalents) of lentiviral vector, approximately 4% of the livercells in the LV-treated groups expressed hFIX, as determined byimmunohistochemistry. Delivery of a lentiviral vector encoding GFPresulted in up to 10% of the hepatocytes expressing FP.

[0115] Vascular delivery of lentiviral vector encoding hFIX in C571B/6mice resulted in a robust humoral response to hFIX. This was alsoaccompanied by a robust anti-VSV-G envelope-directed response. Vascularadministration of lentiviral vectors results in the transduction of thespleen and bone marrow. In addition, transgene expression from acytomegalovirus (CMV) promoter-driven cassette can be readily detectedin splenic antigen-presenting cells, B cells, and to a lesser extent Tcells, following vascular delivery of lentiviral vector. However, thiscan be averted through the use of a liver-specific promoter to drivetransgene expression. This would eliminate the antigen-presentingcell-driven immune response to the lentiviral vector-encoded transgene.

[0116] These results indicate that lentiviral vectors can transduce avariety of cells in the liver to express and secrete the clottingfactor, Factor IX, without apparent hepatocellular cytopathology andwithout requiring cell division. This vector's large coding capacity andits ability to transduce the cells of the liver, the body's largestsecretory organ, supports the use of this vector in the treatment of avariety of genetic diseases.

[0117] Materials and Methods

[0118] Vector constructs and vector production: The LV-hFIX expressionconstruct, pRRL-sin-CMV-hFIX-PRE, a self-inactivating vector, wasconstructed by cloning the hFIX cDNA¹⁵, driven by the CMV IEenhancer/promoter and containing the SV40 poly-adenylation signalsequence, was introduced into a pRRL-based lentivirus transfer vectordescribed previously^(6,7) using standard cloning techniques. Thewoodchuck hepatitis b virus post-transcriptional regulatory element(WPRE) was introduced 5′ of the 3′ LTR of the vector¹⁶.

[0119] The method of production of the 3rd generation lentiviral vectorhas also been described⁶ with some modifications. Briefly, vectors wereproduced by transient transfection of 293T cells. A total of 21.0 μg ofplasmid DNA was used for the transfection of one 10 cm dish: 3.5 μg ofthe envelope plasmid, pMD.G, 5.0 μg of packaging plasmid, pMDL g/p.rre,2.5 μg of pRSV-REV and 10 μg of transfer vector plasmid(pRRL-sin-CMV-hFIX-PRE). After harvesting, vector supernatants werecleared by low speed centrifugation, and filtered through0.22-μm-pore-size cellulose acetate filters. Vectors were thenconcentrated by high speed centrifugation using a SW-28 rotor (Beckman,Fullerton, Calif.) at 19,500 r.p.m. for 140 minutes. Immediately aftercentrifugation, the supernatant was removed and the pellet resuspendedin PBS w/o Ca⁺⁺ and Mg⁺⁺. The vector was then frozen and stored at −80°C.

[0120] Transducing activity was determined in vitro by infecting 1×10⁵HeLa cells using serial dilutions of vector preparations expressingeither hFIX or eGFP in a six-well plate in the presence of Polybrene (8μg/ml; Sigma, St. Louis, Mo.). Following a 16 hour infection, the vectorsupernatant was removed and fresh media was added. Forty eight hourslater, the media was removed again, and fresh media added. After twentyfour hours, the supernatant from cells transduced with LV-hFIX wascollected to measure hFIX expression levels by ELISA. Cells that weretransduced with Lenti-eGFP were collected analyzed by FACS for eGFPtransgene expression. DNA was also extracted from Lentivirus transducedcells and analyzed for the presence of Lentiviral vector genomes asdetermined by TaqMan PCR analyses. Viral p24 antigen concentration wasdetermined by immunocapture (Alliance; DuPont-NEN, Boston, Mass.). Thelentiviral vector preparations had an infectivity of 5×10⁷-1×10⁸ TU/μgp24 Gag antigen.

[0121] Animal procedures: C57BL/6 and NIH Swiss nude mice were obtainedfrom Taconic Laboratories (Germantown, N.Y.) and housed under barrierconditions. 6-8 week old adult mice were administered 100 μl ofLentivirus (4 μg p24) via portal vein or tail vein, intrasplenic ordirect liver. For portal vein injections, cannulations were performed 2days before injection. At various time points following injection, serumwas collected via retro-orbital bleeding. Mice were sacrificed 48 daysfollowing injection. The following tissues were fresh frozen andsubsequently analyzed by TaqMan DNA-PCR or processed for histologicalanalyses: liver, lung, kidney, heart, spleen, brain, pancreas, duodenum,testis and mesenteric lymph node.

[0122] Antigen assay for human Factor IX: Mouse serum was analyzed fortotal human Factor IX antigen by ELISA. Briefly, 96-well plates (Costar)were coated with 2 μg/ml of monoclonal anti-human Factor IX antibody(Boehringer-Mannheim) in 0.1 M carbonate buffer (pH 9.6) at 4° C.overnight. Plates were washed 5× in BBST (BBS containing 0.025%Tween-20). Plates were blocked in 1% (w/v) nonfat dry milk in BBS (89 mMboric acid, 90 mM NaCl, pH 8.3) for 2 hours at room temperature. Sampleswere diluted in 1% (w/v) nonfat dry milk in BBS at 1:4 dilution. 50 μlof sample was incubated on plate for 2 hours at room temperature. Plateswere washed 5× with BBST and incubated with HRP conjugated goatanti-human factor IX antibody (1:100) (Affinity Biologicals, Hamilton,Canada) for 1.5 hours at room temperature. Plates were washed and 50 μlof p-nitrophenyl phosphate substrate solution was added. The reactionwas stopped with 50 μl 2M sulfuric acid and read at 490 nm with amicroplate reader (Molecular Devices, Menlo Park, Calif.). Factor IXlevels were calculated based on a standard curve generated from a serialdilution of purified human Factor IX standard (Calbiochem, La Jolla,Calif.) diluted in 25% control mouse plasma. The values are expressed inng/ml.

[0123] Antibodies to hFIX: Anti-hFIX antibodies were detected by ELISA.Briefly, 96 well plates were coated with human Factor IX (Calbiochem, LaJolla, Calif.). Serum samples were incubated on plate at 1:5 or 1:10dilutions. Horseradish peroxidase conjugated goat anti-mouse IgG wasused as the detection antibody. Plates were developed usingo-Phenylenediamine dihydrochloride (OPD) substrate. The values weredetermined based on a standard curve using a monoclonal anti-humanfactor IX antibody (Boehringer Mannheim, Indianapolis, Ind.).

[0124] Serum Analyses: Serum samples were collected from animals beforeor various time points after gene delivery. Serum glutamine pyruvatetransaminase (sGPT), alkaline phosphatase and creatinine assays wereperformed using kits obtained from Sigma (St. Louis, Mo.). The valuesare expressed in international units/ml.

[0125] Histological analyses: Liver and spleen tissues were collectedfrom the mice at 48 days after administration and fresh frozen. 8 μmsections were cut and stained with hematoxylin and eosin.

[0126] Factor IX Immunohistochemistry: Frozen sections were alsoprocessed for Factor IX immunohistochemistry. Briefly, cryosections werefixed for 15 minutes in 1% paraformaldehyde in PBS, pH 7.4 and washed inPBS with 1% BSA. Sections were blocked with normal donkey serum andwashed in PBS containing 1% BSA. Sections were then incubated with asheep anti-human Factor IX antibody (Affinity Biologicals, Hamilton,Ontario, Canada) at 1:1000 diluted in PBS with 1% BSA. The secondaryantibody used was a biotinylated donkey anti-sheep IgG (JacksonImmunoResearch Laboratories, West Grove, Penn.) (1:3000) followed byincubation with HRP streptavidin complex. All incubation steps were doneat room temperature. Sections were developed with 3′3′ Diaminobenzidine(DAB) substrate. Sections were lightly counterstained with hematoxylin.

[0127] PCNA immunohistochemistry: 8 μm sections were also stained forPCNA proliferation marker. Sections were fixed in 1% paraformaldehyde. Asheep anti-PCNA primary antibody (Santa Cruz Biotechnology, Santa Cruz,Calif.) was used at 1:50 dilution. Washes were in PBS. The rabbitImmunoCruz Staining system (Santa Cruz Biotechnology, Santa Cruz,Calif.) was used for secondary antibody staining with streptavidin-HRPand substrate development. Sections were lightly counterstained withhematoxylin. The slides were viewed using a Zeiss microscope at 20×-100×magnification.

[0128] BrdU-labeling studies: Mice were cannulated at the portal veintwo days before vector administration. Some mice also underwent atwo-thirds partial hepatectomy. Six hours before vector administration,BrdU was delivered to all animals by an Alzet Model 2001 osmotic pump(Durect Corp., Cupertino, Calif.). Subsequently, 200 μl of the BrdUsolution (40 mg/ml in 0.5 M sodium bicarbonate) were added to an Azletpump and then implanted subcutaneously to the mice, resulting in BrdUrelease at 1 μl/h (960 μg BrdU/day). Seven days after vectoradministration, all animals were killed and their livers harvested. BrdUincorporation was detected using a BrdU labeling and detection kit(Roche GmbH, Basel, Switzerland) as described by the manufacturer.

[0129] DNA analyses: DNA was isolated from mouse tissues using theQiagen tissue DNA extraction kit (Valencia, Calif.). 300-500 ng genomicDNA was analyzed by TaqMan PCR using primers specific to the Lentiviraltransfer vector. The primer pair is located upstream of the psipackaging signal in the Lentivirus vector. The following primer set wasused to produce a 64 bp amplicon: forward primer,5′-TGAAAGCGAAAGGGAAACCA-3′ (SEQ ID NO: 1) and reverse primer,5′-CCGTGCGCGCTTCAG-3′ (SEQ ID NO: 2). The probe used was5′-6FAM-AGCTCTCTCGACGCAGGACTCGGC-TAMRA-3′ (SEQ ID NO: 3; AppliedBiosystems, Foster City, Calif.). A final reaction volume of 50 μlconsisted of TaqMan Universal PCR Master Mix; 0.4 μM each primer; 100 nMof FAM probe (Applied Biosystems, Foster City, Calif.). A plasmidcontaining the amplicon sequence was diluted in DNA Hydration buffer andused as a positive control series at 50,000, 5000, 500, 50, 5.0 and 0.5copies per replicate and run in triplicate. Each reaction was run underthe following conditions: 50° C. for 2 minute hold; 95° C. for 10 minutehold; then 40 cycles of 95° C. for 15 seconds and 60° C. for 1 minute inan ABI PRISM 7700 Sequence Detection System unit (Applied Biosystems,Foster City, Calif.). The results were analyzed using the SequenceDetection System (version 1.6.3) software's default settings (baseline3-15; threshold set to 10× standard deviation of the baseline).

[0130] References

[0131] 1. Naldini, L. et al. In vivo gene delivery and stabletransduction of nondividing cells by a lentiviral vector. Science 272,263-7 (1996).

[0132] 2. Naldini, L., Blomer, U., Gage, F., Trono, D. & Verma, I.Efficient transfer, integration, and sustained long-term expression ofthe transgene in adult rat brains injected with a lentiviral vector.Proc Natl Acad Sci USA 93, 11382-8 (1996).

[0133] 3. Naldini, L. Lentiviruses as gene transfer agents for deliveryto non-dividing cells. Curr Opin Biotechnol 9, 457-63 (1998).

[0134] 4. Zufferey, R., Nagy, D., Mandel, R., Naldini, L. & Trono, D.Multiply attenuated lentiviral vector achieves efficient gene deliveryin vivo. Nat Biotechnol 15, 871-5 (1997).

[0135] 5. Blomer, U. et al. Highly efficient and sustained gene transferin adult neurons with a lentivirus vector. J Virol 71, 6641-9 (1997).

[0136] 6. Dull, T. et al. A third-generation lentivirus vector with aconditional packaging system. J Virol 72, 8463-71 (1998).

[0137] 7. Zufferey, R. et al. Self-inactivating lentivirus vector forsafe and efficient in vivo gene delivery. J Virol 72, 9873-80 (1998).

[0138] 8. Bukovsky, A., Song, J. & L, L. N. Interaction of humanimmunodeficiency virus-derived vectors with wild-type virus intransduced cells. J Virol 73, 7087-92 (1999).

[0139] 9. Kafri, T., Praag, H. v., Ouyang, L., Gage, F. & Verma, I. Apackaging cell line for lentivirus vectors. J Virol 73, 576-84 (1999).

[0140] 10. Curran, M., Kaiser, S., Achacoso, P. & Nolan, G. Efficienttransduction of nondividing cells by optimized feline immunodeficiencyvirus vectors. Mol Ther 1, 31-8 (2000).

[0141] 11. Wang, G. et al. Feline immunodeficiency virus vectorspersistently transduce nondividing airway epithelia and correct thecystic fibrosis defect. J Clin Invest 104, R55-62 (1999).

[0142] 12. Wang, X. et al. Efficient and sustained transgene expressionin human corneal cells mediated by a lentiviral vector. Gene Ther 7,196-200 (2000).

[0143] 13. Park, F., Ohashi, K. & Kay, M. Therapeutic levels of humanfactor VIII and IX using HIV-1-based lentiviral vectors in mouse liver.Blood 96, 1173-6 (2000).

[0144] 14. Park, F., Ohashi, K., Chiu, W., Naldini, L. & Kay, M.Efficient lentiviral transduction of liver requires cell cycling invivo. Nat Genet 24, 49-52 (2000).

[0145] 15. Snyder, R. et al. Persistent and therapeutic concentrationsof human factor IX in mice after hepatic gene transfer of recombinantAAV vectors. Nat. Genetics 16, 270-6 (1997).

[0146] 16. Zufferey, R., Donello, J., Trono, D. & Hope, T. Woodchuckhepatitis virus posttranscriptional regulatory element enhancesexpression of transgenes delivered by retroviral vectors. J Virol 73,2886-92 (1999).

[0147] 17. Wion, K., Kelly, D., Summerfield, J., Tuddenham, E. & Lawn,R. Distribution of factor VIII mRNA and antigen in human liver and othertissues. Nature 317, 726-9 (1985).

[0148] 18. Ge, Y., Powell, S., Roey, M. V. & McArthur, J. Factorsinfluencing the development of an anti-FIX immune response followingadministration of AAV-FIX. Blood 97(12), 3733-7 (2001).

[0149] 19. Jooss, K., Yang, Y., Fisher, K. & Wilson, J. Transduction ofdendritic cells by DNA viral vectors directs the immune response totransgene products in muscle fibers. J Virol 72, 4212-23 (1998).

Example 2

[0150] Enhancer1 Augments Expression of Factor VIII in Hepatocytes

[0151] Enhancers are composed of smaller elements that can be combinedalone or with other elements to produce stronger transcription andvaried tissue-specificity. This example describes the creation of anovel liver (hepatocyte) specific synthetic transcriptional enhancerthat is capable of increasing expression in a tissue specific mannerfrom a number of promoter elements, and hence represents a moreclassical “enhancer” that functions with multiple promoters.

[0152] The novel transcriptional enhancer was created by synthesizingoligonucleotide binding sites for six liver (hepatocyte) transcriptionfactors and ligating (linking) these sites together in stoichiometricamounts. The six transcription factors, which are described in theliterature and known to those skilled in the art, are as follows: DBP5′-ATTTATGTAAG-3′; (SEQ ID NO:4) EBP 5′-ATTGCGGCAAT-3′; (SEQ ID NO:5)HNF1 5′-AGGTTAATAATFACCAG-3′; (SEQ ID NO:6) HNF3 5′-GAYTATTTATTYGGCC-3′;(SEQ ID NO:7) HNF4 5′-CGCTGGGCAAAGGTCACCTGCCCCT-3′; (SEQ ID NO:8) andHNF6 5′-AATATTGAYTYGAGGC-3′; (SEQ ID NO:9)

[0153] (where Y represents either C or T) with compatible “sticky” CGCGoverhangs (not shown).

[0154] The random arrays of binding sites were cloned upstream of aminimal human albumin promoter (which has minimal basal transcriptionalactivity on its own) driving expression of GFP in a lentivirus vectorconstruct, thus generating a pool of lentivirus vector plasmids. Viralsupernatants were produced from this plasmid pool and used to transduceHuH7 cells (cell culture hepatocytes) at low multiplicity of infections(MOI). After allowing for expression of the vector, cells were sortedbased on their GFP expression levels using standard fluorescent cellsorting techniques, enriching a population of cells with elevated GFPexpression levels. These cells were grown up as clonal cultures, and DNAwas isolated for amplification of the enhancing element by PCR andsubsequently sequenced.

[0155] These cloned enhancer elements augmented expression levels up totwo orders of magnitude over the enhancer-less vector. The clonedenhancer element described here (clone 1 .1, or Enhancer 1 or Enh1) iscomposed of five types of the six initial elements (EBP, HNF1, HNF3,HNF4, HNF6) in the following order and orientation: HNF1 (sense)-HNF3(sense)-HNF4 (antisense)-HNF1 (antisense)-HNF6 (sense)-EBP(antisense)-HNF4 (antisense). The sequences of Enh1 derived from thehepatocyte elements are in upper case (CGCG sticky ends in italics),with adjacent vector sequences in lower case: 1 gaattcacgc GAGTTAATAATTACCAGCGC GGGCCAAATA AATAATCCGC (SEQ ID NO:10) 51 GAGGGGCAGG TGACGTTTGCCCAGCGCGCG CTGGTAATTA TTAACCTCGC 101 GAATATTGAT TCGAGGCCGC GATTGCCGCAATCGCGAGGG GCAGGTGACC 151 TTTGCCCAGC Gcgcgttcgc cccgccccga tcg.

[0156] The Enhancer1 element was subcloned as an EcoRI-PvuII restrictionfragment (restriction sites underlined) for further manipulation. It isunderstood that variations of these elements with respect to number ofbinding sites, position of binding sites, and orientations of bindingsites will also behave similarly or at elevated levels compared to thisexample. It is also possible that fewer than five of the six types ofelements may be sufficient to generate an enhancing element thatlikewise augments expression.

Example 3

[0157] Enhancer1 Combined with mTTR Promoter Provides High-LevelExpression of Factor VIII in Hepatocytes

[0158] This example, like Example 2 above, addresses the problem ofobtaining adequate gene expression (therapeutic levels) for gene therapybased applications. It also addresses immunological concerns bydirecting expression in a tissue specific manner. Previous constructsused ubiquitous promoters that theoretically would allow gene expressionin antigen presenting cells thereby initiating or acceleratingimmunological activity against the therapeutic molecule.

[0159] This example describes a novel combination of Enhancer1 with ahepatocyte specific promoter derived from the mouse transthyretin (mTTR)gene. A preferred use of this combination involves driving expression ofthe human Factor VIII gene for gene therapy applications using alentivirus-based vector. The novel enhancer element is combined with apromoter element (mTTR) that already contains its own transcriptionalenhancer. The mTTR promoter plus endogenous enhancer has been described(R. H. Costa et al, 1986, Molecular and Cellular Biology 6:4697).

[0160] Enhancer1 was cloned upstream of several (four) different liverspecific promoter elements: mTTR promoter, human alpha-1-antitrypsinpromoter hAAT), human albumin minimal promoter, mouse albumin promoter,and transfected into HuH7 cells for expression analysis. The novelenhancer with the mTTR promoter element (consisting of its own enhancerand promoter) was seen to give the highest levels of expression in thisassay in a cell type restricted manner. This combination of elements wastransferred to the lentivirus system and assayed by transduction ofviral supernatants into HuH7 cells. In this assay, the combination ofnovel enhancer and mTTR promoter was seen to enhance the activity overthe mTTR promoter alone by about two fold. Two reporter genes wereutilized in this analysis: enhanced green fluorescent protein (plasmidand vector pRRLsinpptEnh1mTTR-EGFP) and human factor VIII (plasmid andvector pRRLsinpptEnh1mTTR-hF8(f8/f9)pre). This novel combination of thesynthetic enhancer and the mTTR promoter produced higher levels ofexpression than previous generations of liver specific promoter or“high-level” ubiquitous promoters. This combination of enhancer andpromoter elements can be used to drive therapeutic levels of factor VIIIin vivo.

Example 4

[0161] L-F8 Drives Sustained Expression of Factor VIII in a MouseHemophilia Model

[0162] This example describes a hybrid enhancer and promotercombination, designated L-F8, that can be used to express clottingfactors. The combination is suitable for use with a third generationlentiviral expression system. A restriction map of the vectorpCCLsinL-F8.SMALL.ii.hF8pptpre is shown in FIG. 10.

[0163] The L-F8 hybrid comprises an enhancer (L) and a promoter (F8).The enhancer, L, contains two copies of the a-1-microglobulin/bikuninenhancer (Rouet et al., 1992, J. Biol. Chem. 267:20765-20773; Rouet etal., 1995, Nucleic Acids Res. 23:395-404; Rouet et al., 1998, Biochem.J. 334:577-584; Ill et al., 1997, Blood Coagulation Fibrinolysis8:S23-S30), and was directly cloned from the vectorpCCLsinLSPdxF8pptpre. The promoter used in this hybrid was the humanFactor VIII endogenous promoter. An ˜300 nucleotide fragment, based on apaper that studied the regulation of the Factor VIII promoter(Figueiredo and Brownlee, 1995, J. Biol. Chem. 270:11828-11838), was PCRamplified from genomic DNA and cloned 3′ of the enhancer and 5′ of theFactor VIII coding sequence.

[0164] This vector, as shown in FIG. 10, provides sustained expression(>14 days) in the HemoA null mouse in vivo.

[0165] Throughout this application, various patents and publications arecited. The contents of these patents and publications is incorporatedherein by reference in order to describe more fully the state of theart. In addition, reference is made to techniques commonly understood inthe art. Guidance in the application of such techniques can be found inAusubel et al. eds., 1995, Current Protocols In Molecular Biology, andin Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory Press, NY, the contents of which areincorporated herein by reference.

[0166] The present invention is not to be limited in scope by theembodiments disclosed herein, which are intended as single illustrationsof individual aspects of the invention, and any that are functionallyequivalent are within the scope of the invention. Various modificationsto the models and methods of the invention, in addition to thosedescribed herein, will become apparent to those skilled in the art fromthe foregoing description and teachings, and are similarly intended tofall within the scope of the invention. Such modifications or otherembodiments can be practiced without departing from the true scope andspirit of the invention.

1 10 1 20 DNA Artificial Sequence primer 1 tgaaagcgaa agggaaacca 20 2 15DNA Artificial Sequence primer 2 ccgtgcgcgc ttcag 15 3 24 DNA ArtificialSequence probe 3 agctctctcg acgcaggact cggc 24 4 10 DNA ArtificialSequence transcription factor binding site y = c or t 4 attatgtaag 10 511 DNA Artificial Sequence transcription factor binding site y = c or t5 attgcggcaa t 11 6 17 DNA Artificial Sequence transcription factorbinding site y = c or t 6 aggttaataa ttaccag 17 7 16 DNA ArtificialSequence transcription factor binding site y = c or t 7 gaytatttattyggcc 16 8 25 DNA Artificial Sequence transcription factor binding sitey = c or t 8 cgctgggcaa aggtcacctg cccct 25 9 16 DNA Artificial Sequencetranscription factor binding site y = c or t 9 aatattgayt ygaggc 16 10183 DNA Artificial Sequence enhancer element 10 gaattcacgc gagttaataattaccagcgc gggccaaata aataatccgc gaggggcagg 60 tgacgtttgc ccagcgcgcgctggtaatta ttaacctcgc gaatattgat tcgaggccgc 120 gattgccgca atcgcgaggggcaggtgacc tttgcccagc gcgcgttcgc cccgccccga 180 tcg 183

What is claimed is:
 1. A polynucleotide comprising SEQ ID NO: 5, SEQ IDNO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO:
 9. 2. Thepolynucleotide of claim 1, which comprises SEQ ID NO:
 10. 3. Thepolynucleotide of claim 1, which consists of SEQ ID NO:
 10. 4. Thepolynucleotide of claim 1, further comprising a mouse transthyretin(mTTR) promoter.
 5. The polynucleotide of claim 1, further comprising anα-1-microglobulin/bikunin enhancer and a human factor VIII endogenouspromoter (L-F8).
 6. A transfer vector comprising an expression controlsequence operably linked to a transgene, wherein the expression controlsequence comprises the polynucleotide of claim
 1. 7. The transfer vectorof claim 6, wherein the expression control sequence comprises SEQ ID NO:10.
 8. The transfer vector of claim 6, further comprising a mousetransthyretin (mTTR) promoter.
 9. The transfer vector of claim 6,further comprising an α-1-microglobulin/bikunin enhancer and a humanfactor VIII endogenous promoter (L-F8).